This mentored clinician scientist development award builds upon the candidate's training as a neuropathologist and will enhance her research career development through the study of Hedgehog signaling. Sonic hedgehog (Shh) is a protein that is essential to variety of developmental processes in the central nervous system, limbs, gastrointestinal tract, and other organs. Dysregulation of the Shh pathway has been implicated in human developmental and neoplastic diseases ranging from holoprosencephaly to pancreatic carcinoma. Shh signaling and responses can be broadly conceptualized as the interplay of three major events: the cell-to-cell "diffusion" of Shh, the proliferative effects of Shh, and the Shh-induced differentiation of recipient cells. Critical to normal development is the appropriate spatial and temporal regulation of all three of these aspects of Shh signaling. Recent studies have shown that Shh signals are modulated by heparan sulfate proteoglycans (HSPGs) in a developmentally regulated manner. The aims of this application are to determine the mechanism by which Shh-HSPG interactions alter the Shh response and determine the biologic significance of such interactions at both the cellular and the organismal levels. The candidate hypothesizes that HSPGs modulate Shh signaling by regulating internalization and trafficking of the ligand. To address this question at the cellular level, she will employ a combination of biochemical and genetic approaches to alter Shh-HSPG binding in cultured granule cell neurons and follow downstream consequences on proliferation and specific target gene induction. The candidate will define whether all Shh responses or only a subset rely on HSPG binding, and whether these differences are due to HSPG-mediated changes in Shh trafficking. She will also determine the in vivo significance of Shh-HSPG interactions at the organismal level by examining two newly generated strains of knock-in mice that harbor mutations in the HSPG bindings domain of Shh. The mice will be assessed for patterning defects, regional dysregulation of proliferation and differentiation, and abnormal induction of key target genes. Given that Shh is active in so many tissues and the expression of HSPGs is equally ubiquitous, this project will shed light on a mechanism of growth factor modulation that may have broad applications in development and neoplasia. The Dana-Farber Cancer Institute and Harvard Medical School provide a rich intellectual environment and the necessary resources for the successful training and career development of physician-scientists. The application outlines a comprehensive plan that takes advantage of this unique environment to ensure that the candidate will acquire the knowledge and critical skills to transition to independence.